Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List

Full text release has been delayed at the author's request until August 16, 2025

ETD Abstract Container

Abstract Header

THE ROLE OF PHOSPHORYLATION AS A REGULATORY MECHANISM IN LIQUID-LIQUID PHASE SEPARATION OF TDP-43 LOW COMPLEXITY DOMAIN

Haider, Raza
http://orcid.org/0000-0003-4542-9487
http://rave.ohiolink.edu/etdc/view?acc_num=case1717761693927101

Abstract Details

2024, Doctor of Philosophy, Case Western Reserve University, Physiology and Biophysics.
TDP-43 is a nucleic acid-binding, nucleocytoplasmic protein involved in several cellular processes, including transcription, RNA processing, translation, and stress granule formation. Proteinaceous inclusions made up of misfolded TDP-43 are the hallmark of several neurodegenerative diseases, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and limbic-predominant age-related TDP-43 encephalopathy. Much of the TDP-43 within these inclusions is post-translationally modified, with hyperphosphorylation drawing particular interest as phosphorylated TDP-43 segregates specifically to said inclusions. Like several other RNA-binding proteins associated with neurodegenerative diseases with intrinsically-disordered domains, TDP-43 can undergo liquid-liquid phase separation (LLPS) under physiologic conditions. This process involves the protein self-associating into a reversible, liquid-like droplet phase. While LLPS plays a vital physiologic role in the spatial organization of cellular contents, increasing evidence has linked dysregulated, aberrant phase separation to pathogenesis. In the work described in this thesis, we demonstrated that phosphorylation (modeled via Ser-to-Asp phosphomimetic substitutions) can profoundly affect the LLPS of TDP-43. In the first study, which explored phosphorylation at pathologically-relevant C-terminal sites (residues S403, S404, S409, and S410), we ascertained that phosphorylation changes the forces driving TDP-43 LLPS and imparts a biphasic dependence on NaCl concentration to the protein’s LLPS. This biphasic dependence was due to attractive electrostatic forces tuning hydrophobic forces to drive LLPS at low ionic strengths, and pure hydrophobic forces driving LLPS at high ionic strengths where all electrostatic interactions had been screened out. In the second study, we explored the impact of phosphorylation within the transiently α-helical region (at residues S332 and S333) of TDP-43, a subdomain shown to be crucial for the intermolecular protein-protein interactions that underpin LLPS. Phosphorylation at S332 and S333 has been observed in disease, and we show that phosphomimetic substitutions at these sites severely inhibit LLPS by disrupting the α-helical secondary structure within this subdomain. Furthermore, the α-helical phosphomimetic variants showed slower droplet aging and amyloid aggregation, suggesting that phosphorylation at S332 and S333 may be neuroprotective. Overall, these studies highlight that the effects of phosphorylation on TDP-43 LLPS appear to be site-specific, pointing to the potential regulatory role of this post-translational modification on the protein’s phase separation.
Witold Surewicz (Advisor)
Sudha Chakrapani (Committee Chair)
Matthias Buck (Committee Member)
Ashleigh Schaffer (Committee Member)
Brian Appleby (Committee Member)
James Leverenz (Committee Member)
158 p.
Biophysics
TDP-43; liquid-liquid phase separation; intrinsically-disordered protein; amyloid aggregation; phosphorylation

Recommended Citations

Citations

  • Haider, R. (2024). THE ROLE OF PHOSPHORYLATION AS A REGULATORY MECHANISM IN LIQUID-LIQUID PHASE SEPARATION OF TDP-43 LOW COMPLEXITY DOMAIN [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1717761693927101

    APA Style (7th edition)

  • Haider, Raza. THE ROLE OF PHOSPHORYLATION AS A REGULATORY MECHANISM IN LIQUID-LIQUID PHASE SEPARATION OF TDP-43 LOW COMPLEXITY DOMAIN . 2024. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1717761693927101.

    MLA Style (8th edition)

  • Haider, Raza. "THE ROLE OF PHOSPHORYLATION AS A REGULATORY MECHANISM IN LIQUID-LIQUID PHASE SEPARATION OF TDP-43 LOW COMPLEXITY DOMAIN ." Doctoral dissertation, Case Western Reserve University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=case1717761693927101

    Chicago Manual of Style (17th edition)

case1717761693927101
© 2024, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.